Carcinoma of the lung is the principal cause of cancer deaths in American men. An in vitro model for one of the major histological types, squamous cell carcinoma, has been developed. During long-term culture, these cells become neoplastic while accumulating changes in shape, which are detected by a computerized, morphometric assay. The changes can be reproduced in cells exposed to the tumor promoter, phorbol 12-myristate 13-acetate (PMA), and they can be broken down by a method called factor analysis. One factor each for filopodium (#4) formation and (#12) rounding up, plus several factors caused by endocytosis (#2, #6, #8, #13), have been found. The goal of this research is to analyze the PMA effect in terms of known functions regulated by GTPases of the Rho family. In order to determine which (if any) isozyme of protein kinase C (PKC) is involved in PMA-mediated changes, isozyme-specific anti-sense oligodeoxynucleotides will be put into cultured cells. Trials will be conducted separately with each isozyme, and its level monitored to find out whether it is abolished. The ruffling and shape responses to PMA will then be analyzed. If an isozyme activates a GTPase pathway, a statistically significant difference between factor values for that culture will be observed versus controls treated only with PMA. The studies will reveal whether PKC initiates actin remodeling, and may reveal which GTPase is accessible to the PKC-mediated pathway. For example, analysis of the factor(s) affected will suggest whether filopodia or adhesive structures are targets for PKC. These points of motility regulation are of particular interest, because they are under- expressed in the neoplastic cells. The reason why formation of filopodia and focal adhesion sites is suppressed in neoplastic cells, whereas ruffling is enhanced, will be investigated further through experiments on the interactions amongst GTPase-initiated activities. If PMA-treated cells form more filopodia, this will be detectable through an effect on factor #4's value. If the formation of focal adhesion sites is enhanced by lysophosphatidic acid-mediated rho activation, and this alters the prevalence of filopodia or ruffles, it will be detectable by means of #4 values and #2/#6/#8/#13, respectively. The postulated feedback mechanism accounting for failure of rho- and Cdc42-activated motility will be explored by microinjecting cells with selective inhibitors of these functions, botulinum exoenzyme C3 and anti-Cdc42(GAP) antibody, respectively. The results will further the objective of finding agents that counteract the promoter effect in human bronchial cells and thus, slow the development of cancer.
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